Viral vector complexes having adapters of predefined valence

ABSTRACT

The invention concerns an improvement in the art of inserting and expressing foreign gene into eukaryotic cells. The invention particularly concerns methods and compositions whereby viral vectors can be used to insert and express foreign genes into specifically cells having particular differentiation antigens. A method of determining which differentiation antigens can be used is taught. The invention encompasses complexes of viral particles and adapters that cause the binding and internalization of the vector particles such that a gene of interest in the particle is expressed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 08/363,137, filedDec. 23, 1994 now U.S. Pat. No. 5,753,499, the contents of which areincorporated in its entirety by reference herein.

1. FIELD OF THE INVENTION

The invention involves viral vectors that can be used to transduce atarget cell, i.e., to introduce genetic material into the cell. Thetargets of interest are eukaryotic cells and particularly human cells.The transduction can be done in vivo or in vitro. More particularly theinvention concerns viral vectors, that can be used to transduce one fromamong many types of cell that has a particular acceptor molecule exposedon the target cell's surface.

2. BACKGROUND OF THE INVENTION

A variety of viral based vectors have been employed to transfer and toexpress a gene of interest into a eukaryotic target cell. RecombinantDNA techniques are used to replace one or more of the genes of the viruswith the gene of interest operably linked to a promoter that isfunctional in the target cell. The construct, termed a viral vector,infects the target cell, using the physiological infective “machinery”of the virus, and expresses the gene of interest instead of the viralgenes. Because not all the genes of the virus are present in the vector,infection of the target by the vector does not produce viral particles.Viruses that have been used to infect human or mammalian target cellsinclude herpes virus, adenovirus, adeno associated virus and derivativesof leukemia-type retroviruses. Among the retroviruses of particularinterest in the transduction of cells of human origin are constructsbased on amphotropic retroviruses.

2.1. Use of Amphotropic Retrovirus Vectors

Retroviruses are particularly well suited for transduction of eukaryoticcells. The advantages of a vector based this type of virus include itsintegration into the genome of the target cell so that the progeny ofthe transduced cell express the gene of interest. Secondly, there arewell developed techniques to produce a stock of infectious vectorparticles that do not cause the production of viral particles in thetransduced target cell. Lastly, the production and purification ofstocks vector particles having titers of 10⁶ TCIU/ml can beaccomplished.

One disadvantage of the use of retroviral vectors is that there ispresently no practical general, method whereby a particular tissue orcell type of interest can be specifically transduced. Previous effortsto this end have included surgical procedures to limit to specificorgans the physical distribution of the viral vector particles. Ferry,N., et al., 1991, Proc.Natl.Acad.Sci. 88:8377.

Alternatively, practitioners have taken advantage of the fact that typeC retroviruses only infect dividing cells. Thus, a population of cells,e.g., bone marrow cells, was removed from a subject and cultured ex vivoin the presence of growth factors specific for the specific target cellwhich, thus, comprises most of dividing cells in the culture. See, e.g.,Wilson, J. M., et al., 1990, Proc.Natl.Acad.Sci. 87:439-47; Ohashi, T.,et al., 1992, Proc.Natl.Acad.Sci. 89:11332-36. After transduction thedividing cells must be harvested and, for many purposes, reimplantedinto the subject. The technical difficulties of the ex vivo culturetechnique combined with the unavailability of growth factors of specificfor some types of cells have limited the application of this approach.

A second difficulty presented by the use retroviral based vectors isthat a retroviral particle contains two copies of its genome. There is anonzero possibility of a genetic recombination between the alleles ofthe viral particles. Such recombination can give rise to a replicationcompetent virus that can cause the production of infectious particles bythe target cell. In contrast to herpes virus or adenovirus infectionretroviral infections are not necessarily self-limiting.

Notwithstanding these difficulties retrovirus vectors, based onamphotropic murine leukemia retroviruses that infect human cells, havebeen approved for use in human gene therapy of certain diseases, forexample adenosine deaminase and low density lipoprotein receptordeficiencies and Gaucher's Disease. See, e.g., Miller A. D., 1992,Nature 357:455; Anderson, W. F., 1992, Science 256:808.

One approach to overcoming the limitations of using amphotropicretrovirus vectors in human cells has been to mutate the gene encodingthe protein on the viral surface that determines the specificity ofinfection of the virus, the gp70 protein. Using recombinant DNAtechnology a “mutant” virus is constructed that has had small regions ofthe gp70 sequence replaced by predetermined sequences. The limits ofthis approach are set by the requirement for knowledge of the sequencethat will enable infection of the target of interest. However, when thisknowledge was available, the anticipated alteration in viral specificityhas been observed. Valsesia-Wittmann, S., 1994, J.Virol. 68:4609-19.

2.2. The Use of Viral Vector Complexes to Transduce Target Cells

An alternative to altering the specificity of binding of the gp70protein itself is to employ a second, novel structure that binds or isbonded to both the viral particle and to the target cell. Such novel,independently functioning molecules can be thought of as molecularadapters which, together with the viral particle form a vector complex.In one example of this approach, lactose molecules were covalentlycoupled, by a non-specific reaction, to the envelope proteins of anecotropic retrovirus, which does not normally infect human cells. Ahuman hepatocellular carcinoma that was known to have receptors forlactose-containing proteins was found to be susceptible to transductionby this vector complex, although the integration of the transduced geneof interest in the target cell chromosome was not directly demonstrated.Neda, H., et al., 1991, J.Biol.Chem. 266:14143. No evidence ofexpression was observed in a hepatocellular carcinoma that lacked thelactose specific receptor. The method of Neda results in a variablenumber of binding sites for the exposed acceptor on the target cell,attached to each derivatized or bound envelope protein and, of course,is limited to the case wherein the target cell has a lactose receptor.

Another approach to the use of adapter molecules involved an adapterthat was not covalently coupled to the vector. The use of this type ofadapter has been attempted by Roux and his colleagues, who havepublished several reports that relate to this strategy. PatentPublication FR 2,649,119 to Piecheczyk, Jan. 4, 1991; Roux P., et al.,1989, Proc. Natl. Acad. Sci. 86:9079-83; Etienne-Julan, M., et al.,1992, J. Gen. Virol. 73:3251-55. Roux and colleagues have constructedadapters from two types of proteins, both typically antibodies, bybiotinylating the proteins and utilizing avidin or streptavidintetramer, a protein which binds four biotin molecules, to formaggregates of up to four of the biotinylated proteins. The first type ofproteins was an anti-gp70 antibody, which binds to the viral particle.The second type of protein was one of a variety of proteins thatspecifically bind to the target cells and could be an antibody or otherprotein. The adapter of Roux contained a streptavidin tetramer and fourother protein molecules. Each adapter contained one streptavidintetramer, but the aggregates were otherwise random, i.e., all possiblecombinations of the two other types of proteins were possible includingaggregates having only anti-gp70 and only the target cell specificprotein.

To offset the difficulties attendant with the use of mixtures ofrandomly aggregated proteins, Roux did not employ pre-formed adaptersbut rather constructed the adapters, in situ, i.e., on the surface ofthe target cell, by successively exposing the cells to the target cellspecific protein, the streptavidin, the anti-gp70 and lastly to theviral vector itself. Even when constructed in situ, the adaptermolecules of Roux consisted of a random mixture having no predeterminednumber of viral or target cell binding sites.

To allow for the completion of this multistep process the target cellsmust be prevented, by some means, from internalizing the components ofthe aggregates prior to their completion. The method adopted by Roux wasto reduce the temperature of the culture. Thus, the work of Roux doesnot yield a system that can be used at all in an in vivo setting. Evenex vivo, the complex of adapter and vector must be constructed in amultistep process during which the metabolism of the target cell must beinhibited.

3. SUMMARY OF THE INVENTION

The invention concerns the use of viral vectors that have proteins onthe surface of the viral particles, hereinafter termed envelopeproteins, that do not bind to the target cell of interest. The inventioninvolves a new type of adapter that provides a fixed, predefinedvalence, i.e., number of binding sites, specific for an exposed acceptormolecule on the target cell, on each envelope protein molecule havingsuch an adapter. Because the binding sites for the viral particle andthe target cell are different, the adapters of the invention can beconstructed in the absence of the target cell. The preformed complexesof adapters and viral particles that can then be used to transduce agene of interest into the target cell.

In one embodiment, the invention comprises a complex of a viral vectorand a non-covalently bonded difunctional molecule, i.e, a moleculehaving at least one site for linking with the virus and a predefinednumber of binding sites specific for the target cell of interest. In aparticular embodiment of this type, the difunctional molecule has asingle binding site for the virus and a single site for the target cell.

The invention also encompasses complexes comprising a viral vector inwhich some or all of the envelope protein molecules of the virus aremodified by formation a new covalent bond. There are three differentembodiments of this form of the invention.

Firstly, the adapter molecule can be a binding polypeptide that replacesat least about 25 amino acids of the envelope protein of the virus andwhich, thereby, forms a fusion protein. Such fusion proteins can be madeby linking, through recombinant DNA techniques, the fragment of the geneencoding the fragment of the envelope protein to a fragment of a genethat encodes a parent protein having the same, desired bindingspecificity as the binding polypeptide. As used herein, a fusion proteinis a protein having at least two blocks of contiguous sequence, of about10 or more amino acids in length, that are derived from two differentparent proteins. In a preferred embodiment, the viral particle containsa mixture of normal envelope proteins and fusion proteins.

Secondly the adapter can consist of a linking molecule and a polypeptideof an envelope protein/polypeptide fusion protein, as described above.The non-envelope polypeptide of the fusion protein is complementary tothe linking molecule and is, hence, termed a linking polypeptide. Inthis embodiment, the linking molecule is not covalently bonded to thevector. Rather, the linking molecule is itself difunctional. It containsa ligand functionality, which is complementary to the linkingpolypeptide, affixed to the viral surface, and an acceptor bindingportion which contains a predefined number of binding sites for anexposed acceptor on the target cell surface.

The third embodiment employs the linking molecule, as described above,but does not utilize an envelope protein/linking polypeptide fusionprotein. Rather in this embodiment a linking site is covalently affixedto the envelope protein after the vector particle has been constructed.

The invention further provides methods whereby a one skilled in the artcan determine whether a particular antibody is suitable for use as atarget cell specific protein and encompasses methods of use of thepreformed viral vector complexes.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. FACS histogram of β-galactosidase-dependent fluorescent stainingof SupT1; A: untransduced; (B,C): transduced with aψ-2BAG/SA-PA/anti-CD4 vector complex in two separate experiments.

FIG. 2. Mean β-gal fluorescent staining of CD4⁺ (stippled fill) and ofB220⁺ (diagonal fill) cells recovered from a transduced mouse in vivo:A: ψ-2BAG/SA-PA/anti-CD4 vector complex; B: ψ-2BAG/SA-PA only; C:ψ-2BAG; D: without viral particles. Results of two experimental animalsare shown.

FIG. 3. Time course of the internalization of vector complexes. Daudi,anti-HLA-DR antibody only (-∘-); SupT1, anti-CD4 antibody only (-▪-);Daudi, vector complex (-▴-); SupT1, vector complex (-□-).

FIG. 4. Schematic of the gene encoding a streptavidin/env fusion proteinand the operably linked cytomegalovirus promoter (CMV-pro) as found inpST-env.

FIGS. 5A and 5B. FACS histograms of β-gal fluorescent staining oftransduced Daudi cells. FIG. 5A: ψ-2BAG (negative control). FIG. 5B:pST-env-ψ-2BAG/biotinylated anti-HLA-DR vector complex. Note thepresence of cells in channels >100 in FIG. 5B only.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a means for modifying the genome ofeukaryotic cells, such as mammalian cells or avian cells, and, moreparticularly, of human cells for medical practice and also of the cellsof domesticated animals that are valuable for agriculture andrecreational purposes for veterinary practice. The invention providesfor the introduction and expression of genetic material into the cellsby means of a viral vector complex. In the viral vector, some or all ofthe viral genes have been replaced by a gene that is to be expressed inthe eukaryotic target cell. The essential viral genes that have beenremoved from the vector are, in general, inserted into the genome of thecell line that is used to produce stocks of the viral particles. Theproducer cells lines thus complement the defects that are present in theviral vector. In some embodiments, the only viral gene contained in thegenome of the vector is a gene that is needed for the packaging of thevector genome into the viral particles.

The construction of viral-based vectors suitable for the generalexpression of genes in cells that are susceptible to infection by thevirus is described the following patent publications: WO 89/05345 toMulligan, R. C. and others, WO 92/07943 to Guild, B. C. and othersconcerning retroviral vectors; WO 90/09441 and WO 92/07945 to Geller, A.I. and others concerning herpes vectors; WO 94/08026 to Kahn, A. andothers, and WO 94/10322 to Herz, J. and others concerning adeno virusvectors; and U.S. Pat. No. 5,354,678 to Lebkowski and U.S. Pat. No.5,139,941 to Muzcyzka concerning adeno-associated virus. Packagingsystems for the production of retroviral vectors have been described byDanos, O., and Mulligan, R. C., 1988, Proc.Natl.Acad.Sci. 85:6460-64,and by Landau, N. R., & Litmann, D. R., 1992, J.Virol. 66:5110-13.

The present invention is an improvement in the viral vector art. Theinvention replaces the viral vector with a vector complex which consistsof two portions: a viral portion and a target cell specific portiontermed an adapter. The adapter subserves the function of binding thevector to the target cell in such a way that the vector is internalizedinto the cell. This function is necessary for infection and expressionof the gene of interest. Without limitation as to theory, the inventionaccomplishes two improvements by providing an adapter portion. Firstly,the use of an adapter allows the vector to be constructed so that it isunable to infect the target cell, or any cell from the same species, inthe absence the adapter. In certain embodiments of the invention, thevector is based on a virus that is normally unable to infect the targetspecies and the adapter is not encoded by a gene in the vector. Sinceinfection is possible only when the adapter is present, a recombinantvirus that causes an inadvertent, iatrogenic infection becomes virtuallyimpossible.

Secondly, the adapter can be provided with a variety of specificities.The application discloses methods of constructing an adapter comprisingan antibody specific for an acceptor on the target cell or an antigenbinding fragment of the antibody, so long as the acceptor and attachedvector complex are internalized into the target cell after the vectorcomplex is bound. Thus, there are a large number of cell surfaceantigens suitable for use as acceptors and for which antibodies arealready available. Such structures include, but are not limited to, theclass I and class II Major Histocompatibility Antigens; receptors for avariety of cytokines and cell-type specific growth hormones,interleukins, interferons, fibroblast growth factors, erythropoietin,transforming growth factors, tumor necrosis factors, colony stimulatingfactors and epidermal growth factor; cell adhesion molecules; transportmolecules for metabolites such as amino acids; the antigen receptors ofB- and T-lymphocytes; and receptors for lipoproteins. The inventionmakes possible the specific infection of a cell type by allowing theemploy of differentiation antigens as acceptors for the viral vectorcomplex.

The invention is used to transduce a gene of interest into a targetcell. In practicing the preferred embodiment of the invention, thecomplex of the viral vector and the adapter is formed prior to theinteraction of the adaptor or of any part of the adaptor and the targetcell acceptor.

The practice of the invention can be performed by culturing the targetcells ex vivo. The cultured cells can be continued in culture to producethe product encoded by the transduced gene. Alternatively, the ex vivotransduced cell can be implanted into a subject, which can be the hostfrom which the cultured cells were obtained.

In a yet further embodiment, the viral vector complex can beadministered directly to the subject thereby obviating the need for anyex vivo cell culture. The routes of administration to the subject can beany route that results in contact between the vector complex and thetarget cell. Thus for example, intravenous administration is suitablefor target cells in the hepatic, splenic, renal cardiac and circulatoryor hematopoietic systems. The vector complex can also be administered bycatheterization of the artery or vein leading to the target organ,thereby allowing the localized administration of the complex. Thecomplex can also be administered by inspiration when the target cellsare in the respiratory system.

Genes that can be transduced by the practice of the invention includeany gene that can be expressed in a eukaryotic system. Illustrativeexamples of genes that can be expressed by use of the present inventioninclude glucocerebrosidase, adenosine deaminase, and blood coagulationfactors such as factor VIII and factor IX.

The viral component of the vector complex can be based on any virus, theparticles of which are unable to bind or have been modified to be unableto bind to cells of the same species as the target cell. A non-limitingexample of the first type of virus are the murine ecotropic leukemiaretrovirus viruses, e.g., Moloney Leukemia Virus or AKV. Alternatively,chemically modified viral particles can be employed. By way of anon-limiting example, a viral particle can be biotinylated by any methodthat results in the extensive covalent attachment of the biotinmolecules to the envelope protein of the virus. Such extensivebiotinylation blocks the function of the envelope protein, therebymaking the virus non-infectious in the absence of an adapter having aligand portion complementary to a linking site on the viral particle. Inthis embodiment the biotin itself provides the linking site. In additionto ecotropic retroviruses, viruses that can be employed to constructvectors according to this embodiment of the invention includeamphotropic retrovirus, herpes virus, adenovirus and adeno-associatedvirus.

The embodiments of the invention are described in greater detailhereinafter.

5.1. The Complex of an Unmodified Virus Particle and an Adapter

In one embodiment the vector complex consists of a vector having one ormore envelope proteins that are substantially identical to the virionsurface protein or proteins of the parent virus. As used herein, unlessotherwise indicated, an envelope protein of a virus is any protein of aviral particle accessible to macromolecules, i.e., an exposed surfaceprotein. A viral envelope protein is an envelope protein which is anon-artifactual constituent of a virus, e.g., the product of the envgene of an ecotropic retrovirus.

The adapter is a molecule, not covalently bonded to the viral vectorparticle, that has two functionalities. The first functionality consistsof a defined number of sites that specifically and stably bind to anenvelope protein of the particle. This functionality of the adapter canbe a peptide, an oligonucleotide, an antibody or an antigen bindingfragment of an antibody, or a polypeptide derived from an envelopebinding domain of the natural receptor of the virus or a homolog of anatural receptor. The second functionality consists of a defined numberof binding sites that specifically and stably bind to an acceptormolecule that is exposed on the target cells surface. In a preferredembodiment the two functionalities are synthesized as distinct portionsof a single polymer. When the functionalities are polypeptides, theadapter is a fusion protein.

An illustrative specific embodiment of the invention is provided inco-pending commonly assigned, U.S. patent application Ser. No.08/132,990, filed Oct. 7, 1993, which is hereby incorporated byreference. In this embodiment, the envelope binding function issubserved by a fragment of a protein that is the human homolog of themurine ecotropic virus receptor, which has been further modified so thatit binds to the ecotropic virus gp70. The target cell binding functionis subserved by the antigen binding fragment from the monoclonalantibody B3 that binds to a carbohydrate moiety on a human mucinouscarcinoma. Pastan, I., et al., 1991, Cancer Research 51:3781.

5.2. The Complex of a Modified Viral Vector and an Adapter

5.2.1. The Modification of the Envelope Proteins

The modification of the viral vector can be performed either before orafter the producer cell line is established. When the modification isperformed after the producer cell is established, the producer cell linesupernatant is concentrated and the viral particles are then furtherpurified by gel filtration or sucrose density gradient centrifugation,following techniques known to those skilled in the art. Afterpurification the viral particles can be modified by any chemicaltechnique which causes the formation of covalent bonds in proteinswithout causing their denaturation or causing the disruption of thelipid membrane of the virus in the case of enveloped viruses. In apreferred embodiment the chemical modification is performed by thereaction of a biotin-N-hydroxysuccinimide (biotin-NHS) with the virus.Although this reaction renders the virus non-infectious, absent anadapter, such viral preparations are, nonetheless, suitable for thepractice of the present invention.

Other techniques of chemical modification of the viral particles thatcan be used to practice the invention include the use ofphotoactivatable reagents, e.g., N-(4′-azido-nitrophenylamino) grouplinked to biotin (PHOTOBIOTIN), and the use of an activated disulfide,i.e., dithio-2-pyridyl containing compound, to modify the envelopeproteins of the viral particles.

In an alternative embodiment the invention can be practiced bymodification of a viral envelope protein, a non-limiting example beingthe env gene of a murine ecotropic retrovirus, prior to the productionof viral particles in the producer line. According to this embodiment ofthe invention a fusion protein is constructed by recombinant DNAtechnology. The recombinant gene encoding the fusion protein is thentransfected into a producer cell line. In one embodiment, the envelopeprotein of the vector does not bind to the cells of the target cellspecies, genes encoding both the normal envelope protein and a fusionprotein containing a fragment of the envelope protein can be present inthe producer line. In the alternative embodiment wherein the completeenvelope protein of the vector bind to such cells, the envelope proteingene is replaced by the envelope protein/binding peptide fusion proteingene.

The envelope protein portion of the fusion protein is selected so thatthe fusion protein is incorporated into the lipid membrane ofencapsulated (enveloped) viral particles or into the outer surface ofthe capsid of encapsidated viruses. The binding polypeptide is selectedto bind to a linking molecule, i.e., to provide a “linking site”. Thestability and suitability of any given candidate fusion protein can bereadily determined, once the gene encoding it is constructed. The geneencoding the candidate fusion protein can be transfected into a suitablecell line, e.g., an NIH 3T3 cell, and the expression on the transfectedcells' surface of a functional fusion protein can be determined byexposure to a complementary ligand portion of the linking molecule,complexed with a reporter molecule such as a fluorescent tag. In anon-limiting example the binding polypeptide is a fragment ofstreptavidin encoding residues 16-133 of the streptavidin protein andthe remainder of the protein is derived from the env gene of the AKRVirus. Two fragments of a gene encoding a envelope protein are used inthe example: a 507 bp fragment extending from the U5 region andincluding the env protein leader, extending from residue Met⁻⁴⁹ toresidue Pro⁻¹ and a 1323 bp fragment that encodes residues Gly²²³ to thecarboxy terminal Glu⁶³⁸ of the env protein. The sequence numbering ofLenz, J., et al., 1982, J.Virol. 42:519, which is hereby incorporated byreference, is used. The resultant viral vector has a linking site thatis complementary to a biotin molecule.

5.2.2. The Linking Molecule

The linking molecule can be any molecule that has functionalities thatspecifically and stably bind to, firstly, the linking site, provided onthe envelope protein of the vector particle and, secondly, to theexposed acceptor on the target cell. The functionalities are termedhereinafter, respectively, the “ligand portion” or “ligand” and thetarget cell “acceptor binding portion” or “acceptor binder”. It isnecessary for the invention that the functionalities be different, i.e.,that the ligand portion does not form a stable complex with the acceptornor the does the acceptor binder form a stable complex with the linkingsite. The complex can be preformed most readily when there are definednumbers of both types of functionalities. The number of predefined sitesof the acceptor binding functionality is not critical to the invention.The number can be one or more than one. For use in vivo it is preferredthat the number of acceptor binding functionalities on each linkingmolecule be small, most preferably between one and four. The number ofthe ligand portion functionalities on each a linking molecule is notcritical and such number need not be predefined and invariable among thelinking molecules.

Non-limiting illustrative examples of linking molecules are as follows:

When the linking site is a biotin binding functionality, the ligandportion can be biotin. The linking molecule can be a biotinylatedantibody, a biotinylated cytokine or growth factor or the like. Thedegree of biotinylation is not critical so long as the acceptor bindingfunction is not inhibited. The number of biotin molecules required toform an effective vector complex is not critical. Variation of the timeof the reaction and concentration of the reagents used to biotinylate anacceptor binder can be employed to determine the optimal number ofbiotins per linking molecule so that the formation of vector complexesoccurs in a convenient period of time and at practically attainableconcentrations of viral particles.

Alternatively, the linking site introduced into the viral particle canbe a biotin. In this embodiment the ligand portion of linking moleculecan be comprised of a streptavidin or a streptavidin fragment. When theacceptor binding portion of the linker molecule is comprised of anantibody, the linking molecule can be comprised of astreptavidin/Protein A fusion protein an example of which is disclosedand claimed in U.S. Pat. No. 5,328,985 and any antibody for a suitableexposed target cell acceptor.

6. EXAMPLES 6.1. Example 1

An Adapter that is Intended to function with an Unmodified Virus

An example of a fusion protein that functions as a soluble adapter foran unmodified ecotropic retrovirus vector is provided by the fusionprotein B3(Fv)-Ex3mH13. This fusion protein is based on the B3(Fv)-PE40fusion protein which is a fusion protein between an antigen bindingfragment B3(Fv) and a fragment of an exotoxin. The B3 monoclonalantibody is specific for a carbohydrate antigen present on a mucinousadenocarcinoma and the antigen binding fragment has a similar bindingspecificity. The PE40 fragment is replaced by the third external domain,residues 210-249 of the human protein H13, which is the human homolog ofthe murine ecotropic retroviral receptor. In Ex3mH13 the amino acid ofthe H13 protein at positions 239, 240, 242 and 244 of H13 have beenreplaced by the amino acid at the homologous position of the murineecotropic retroviral receptor, so that the Ex3mH13 can be non-covalentlyaffixed to the viral particle by interaction with the gp70 envelopeprotein of the virus.

The construction of the expression vector, pBH3, which can be used toexpress the B3(Fv)-Ex3mH13 fusion protein in E. coli is described indetail Example XI of co-pending, commonly assigned U.S. patentapplication Ser. No. 08/132,990, filed Oct. 7, 1993, which is herewithincorporated by reference and in patent publication WO 93/25682 toMeruelo and Yoshimoto, published Dec. 23, 1993. The plasmid pBH3 can beused to express the B3(Fv)-Ex3mH13 fusion protein. The plasmidpTGFA/H13, which can be used to express a fusion protein comprisingresidues 1-50 of TGF_(α)and the above-described Ex3mH13 domain has beendeposited in the ATCC on Dec. 22, 1994 as ATCC No. 69728.

6.2. Example 2

An Adapter Comprising a Modified Envelope Protein, a Linking Moleculeand an Anti-MHC Antibody

6.2.1. Materials and Methods

Viruses and cell lines. All cells used in the current experiments wereobtained from the ATCC except for SupT1, which was generously providedby Dr. J. Sodroski (Dana Farber Cancer Institute, Boston, Mass.), andmaintained in our laboratory at NYU Medical Center. Producer cell lines,ψ-2BAG (ATCC NO. CRL 9560) and NIH 3T3, Daudi and SupT1 cells weremaintained in either Dulbecco's modified Eagle's medium (DMEM) orRPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum(FBS), and a 1% antibiotic/antifungal solution at a final concentrationof 100 IU penicillin and 1% fungizone. The subculture procedure involvesremoval of medium, treatment with fresh trypsin (0.25%) solution for 2-3min, rising and removal of the trypsin. The cells are then incubatedfurther until they detach, then fresh medium is added. The cells areaspirated and dispensed into new flasks at a ratio of 1:4 to 1:20. Cellswere routinely propagated in culture in a standard humidified incubatorat 37° C. in a 5% CO₂/95% air atmosphere. Falcon (Becton Dickinson,Lincoln Park, N.J.) plastic tissue culture dishes were obtained fromFisher Scientific (Springfield, N.J.). Viruses were periodically testedfor acquisition of inappropriate host range by recombination.

Viral purification and concentration. Vector supernatants from producercell lines were concentrated using the Millipore Pellicon tangentialflow filtration system (Millipore, Bedford, Mass.) with a PLMK000C5cassette (5 square feet, 300,000 NMWL). A pump was used to exert a lowmembrane feed pressure of 5 psi. Concentration was achieved within 30min. After concentration the material was aliquoted in 20 ml samples andkept frozen at −70° C. until further use. For purification, 20 ml ofconcentrated material was either passed through a Sepharose 4BCL(Pharmacia) column or banded through sucrose gradients. For columnpurification, the Sepharose 4BCL column is pre-equilibrated withphosphate buffered saline (PBS). After loading the material is elutedwith PBS and fractions assayed for O. D. at 260 nm. Peak fractions werechecked for virus by Western blot using goat anti-gp70 antibodies andrabbit anti-goat alkaline phosphatase. Sucrose density gradientpurification was done as previously described (Bach, R., & Meruelo, D.,1984, J. Exp. Med. 160:270).

Bacterial strains and plasmids. E. coli strains HMS171 (Campbell, J. L.,et al., 1978, Proc. Natl. Acad. Sci., U.S.A. 75:2276-2280) and DH5a(Sambrook, J., et al., 1989, Molecular Cloning: A Laboratory Manual, 2nded. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) wereused for cloning. Lysogen BL21(DE3) (Studier, F. W., & Moffat, B. A.,1986, J. Mol. Biol. 189:113-130; Studier, F. W., et al., 1990, MethodsEnzymol. 185:60-89) was used for expression. This lysogen carries thecloned T7 RNA polymerase gene in the chromosome under the lacUV5promoter. pTSA-18F is an expression vector for streptavidin-containingchimeric proteins (Sano, T., & Cantor, C. R., 1991, Biochem. Biophys.Res. Comm. 176:571-577). This plasmid carries the DNA sequence for aminoacid residues 16-133 of mature streptavidin, followed by a polylinkerregion, under the T7 promoter ø10. pR1T11 (Löwenadler, B. et al., 1986,EMBO J. 5:2393-2398) carries the protein A gene (Uhlén, M., et al.,1984, J. Biol. Chem. 259:1695-1702) corresponding to the signal peptide(Region S) and the five IgG-binding domains (Regions E, D, A, B, and C),followed by a polylinker placed in the sequence for the cell wallattachment domain (Region X). pLyS carrying the cloned T7 lysozyme genewas used to reduce the basal level of T7 RNA polymerase activity in thehost cells (Studier, F. W., & Moffat, B. A., 1986, J. Mol. Biol.189:113-130; Studier, F. W., et al., 1990, Methods Enzymol. 185:60-89)

Construction of expression vectors. pTSAPA-2 was kindly provided by Drs.Takeshi Sano and Charles Cantor. The construction and use of pTSAPA-2can be found in U.S. Pat. No. 5,328,985. Sano and Cantor constructed thepTSAPA-2 vector by inserting a part of the protein A gene into anexpression vector for streptavidin-containing chimeric proteinspTSA-18F. pTSAPA-2 was constructed by inserting a 490 bp Rsa I-Hind IIIfragment of pR1T11 (filled-in using DNA polymerase I large fragment)encoding two IgG-binding domains (Regions E and D) into the Sma I andBamH I (filled-in) sites of pTSA-18F.

Expression of streptavidin-Protein A chimeric proteins. Expression ofthe gene fusion of streptavidin with protein A was carried outessentially according to the method previously described (Sano, T., &Cantor, C. R. 1990, Proc. Natl. Acad. Sci. U.S.A. 87:142-146; Sano, T.,& Cantor, C. R. Biochem. Biophys, Res. Comm. 176:571-577). LysogenBL21(DE3)(pLysS) was obtained from Novagen. It was transformed with anexpression vector and grown at 37° C. with shaking in LB mediumsupplemented with 1 mM MgSO₄, 0.2% glucose, 1.5 μM thiamine, 0.5%casamino acids (Difco Laboratories), 2 μg/ml biotin, 150 μg/mlampicillin and 34 μg/ml chloramphenicol. When the absorbance at 595 nmof the culture reached 0.6, 100 mM isopropyl-β-D-thiogalactopyranosidedissolved in water was added to a final concentration of 0.5 mM toinduce the T7 RNA polymerase gene placed under the lacUV5 promoter.After the induction, the cells were incubated at 37° C. with shaking.Expression in minimal medium had the advantage that proteolysis of theexpressed chimeric protein was substantially reduced.

Purification of streptavidin-protein A chimeric protein. Purification ofthe expressed streptavidin-protein A chimeric protein was carried out at4° C. or on ice, unless otherwise stated. The culture (100 ml) ofBL21(DE3)(LysS)(pTSAPA-2) incubated for 2 hours after the induction wascentrifuged at 2,900×g for 15 min. The cell pellet was suspended in 10ml of 2 mM EDTA, 30 mM Tris-Cl (pH 8.0), 0.1% Triton X-100, 0.5 mM PMSFto lyse the cells and the lysate was stored frozen at −70° C. untilused. To the thawed cell lysate, PMSF, leupeptin, and pepstatin A wereadded to a final concentrations of 0.5 mM, 1 μM, and 1 μM, respectively.The addition of the proteinase inhibitors reduced degradation of theexpressed chimeric protein at early stages of the purification. Thelysate was then treated with 10 μg/ml deoxyribonuclease I and 10 μg/mlribonuclease A in the presence of 12 mM MgSO₄ at room temperature (≈20°C.) for 20 min. The mixture was centrifuged at 39,000×g for 15 min, andthe precipitate was dissolved in approximately 100 ml of 7 M guanidinehydrochloride. The solution was dialyzed against 150 mM NaCl, 50 mMTris-Cl (pH 7.5), 0.05% Tween 20, 0.1 mM PMSF, 1 μM leupeptin, 1 μMpepstatin A, 0.02% NaN₃. To achieve slow removal of guanidinehydrochloride, the dialysis bag containing the protein solution was leftovernight in the dialysis solution (≈1,000 ml) without stirring,followed by several changes of the dialysis solution and dialysis withstirring. The dialysate was centrifuged at 39,000×g for 15 min, and thesupernatant was applied to an IgG Sepharose 6 Fast Flow column (1.2×1.1cm) previously equilibrated with 150 mM NaCl, 50 mM Tris-Cl (pH 7.5),0.05% Tween 20. Unbound proteins were removed by washing the column withthe same solution, and the column was washed with 2.5 ml of 5 mMammonium acetate (pH 5.0). The bound protein was eluted with 0.5 Macetic acid adjusted pH to 3.4 with ammonium acetate, and dialyzedagainst 1.0 M NaCl, 50 mM sodium carbonate (pH 11.0). The dialysate wasclarified by centrifugation at 39,000×g for 15 min. Each fraction wasthen tested for activity by ELISA. The activity was found to elute infractions (1 ml each) one through five, which were pooled and applied toa 2-Iminobiotin agarose (24) column (1.2×1.2 cm) previously equilibratedwith the 10 bed volumes of 1M NaCl, 50 mM NaCHO₃ (pH 11.0). Afterunbound proteins were removed with 1M NaCl, 50 mM NaCHO₃ (pH 11.0), thebound proteins were eluted with 6M urea, 50 mM ammonium acetate (pH4.0). The eluted proteins were dialyzed 3 times against Tris-bufferedsaline (TBS; 150 mM NaCl, 20 mM Tris-Cl (pH 7.5)) containing 0.02% NaN₃,and the dialysate was stored at 4° C. after filtration through a 0.22 mmfilter (Millex-GV, Millipore). Each fraction was again tested foractivity by ELISA.

Viral infectivity assays and titering. On day 1 NIH3T3 cells were seededat 1×10⁵ cells/well of a six-well tissue culture plate (BectonDickinson, Lincoln Park, N.J.) and incubated at 37° C. in 5% CO₂. On day2, serial ten-fold dilutions of vector specimen in medium containing 8μg/ml Polybrene were added to the target cells and incubated at 37° C.for an additional 4 hr, after which the medium was removed and replacedwith regular medium containing 2 μg/ml Polybrene. Cells were assayed forX-gal activity 48 hr later. Vector titer was calculated as the number ofcolony forming units (cfu) per ml. For5-bromo-4-chloro-3-indolyl-β-D-galactosidase (Sigma) (X-gal)visualization of β-galactosidase (β-gal) activity in intact cells, themethod of Dannenberg and Suga (Methods for Studying MononuclearPhagocytes, Ed. by Adams, D. O., et al., (Academic Press, New York), pp.375-396, 1981) was used, following fixation in 0.5% glutaraldehyde for15 min.

Determination of concentration of proteins. To determine proteinconcentrations we used the standard and micro assay proceduresrecommended by the Bio-Rad. For the standard procedure, we preparedseveral dilutions of protein standard (supplied) containing from 0.2 toabout 1.4 mg/ml. We then placed 0.1 ml of standard and appropriatelydiluted samples in clean, dry test tubes. We placed 0.1 ml sample bufferin “blank” test tube. We added 5.0 ml of diluted dye reagent (provided),vortex, and after 5 minutes to one hour, measured the OD₅₉₅ versus thereagent blank. These OD₅₉₅ values were plotted versus concentration ofstandards and the unknown values were determined from the standardcurve. For the microassay procedure, protein standard from 1 to 25 μg/mlwere used, and 0.8 ml of appropriately diluted samples were used with0.2 ml of dye reagent concentrate. Both procedures are otherwise thesame.

Biotinylation of viruses. Biotin-N-Hydroxysuccinimide ester (biotin-NHS)(biotin-X-NHS, Calbiochem, Catalog No. 203187) was used to biotinylatethe envelope proteins. Biotin-X-NHS introduces a spacer between thebiotin and target ligand, reducing steric hindrance that can diminishthe efficiency of streptavidin binding. For this procedure, 1 mg ofvirus were dissolved in 0.9 ml of sterile distilled water. A 0.1 ml of a10×Buffer (NaHCO₃) was added and vortexed. Freshly prepared biotin wasused (11 mg Biotin-X-NHS ester in 0.25 ml dimethylformamide) at a 0.1 Mconcentration. This biotin solution was added to the equilibratedprotein solution and incubated at room temperature for one hour withgentle agitation using a rocker. At the end of the hour theprotein-biotin solution was dialyzed several times against 0.01 M PBS,pH 7.3, to remove unreacted biotin-X-NHS ester. The biotin-labeledprotein solution was stored at 4° C. until further use.

FluoReporter lacZ Flow cytometry. We chose to use a fluoreporter “lacZ”flow cytometry assay for the detection of lacZ-β-Galactosidase in singlecells developed from Molecular Probes, Inc. (Eugene, Oreg.).

Chromogenic β-galactosidase substrates such aso-nitrophenyl-β-D-galactopyranoside (ONPG) and5-bromo-4-chloro-3-indolyl galactoside (X-gal) have been widely used tomonitor β-galactosidase activity resulting from lacZ gene expression.However, chromogenic lacZ assays are relatively insensitive and requirethe use of bulk cell extracts. More recently, measurements of thefluorescence resulting from enzymatic cleavage of fluoresceindi-β-D-galactopyranoside (FDG) has been utilized to develop a lacZ assaybased on fluorescence-activated cell sorting analysis which is at leasteight orders of magnitude more sensitive than chromogenic methods.Another benefit of the method is that it permits analysis of individualcells in a population by use of the FACS analyzer.

All reagents were obtained from the manufacturer and used as recommendedby the manufacturer. Essentially, in this method a cell suspension isprepared from exponentially growing cells and the substrate FDG isloaded into the cells at 37° C. by hypotonic shock. The loading processis terminated by dilution of the cells into ice-cold isotonic media. Atthis low temperature, the cell membrane is relatively impermeable to thesubstrate and hydrolysis results. With the substrate and products lockedinside each cell, the rate of hydrolysis of the substrate to fluoresceinis monotonicly related to the concentration of β-galactosidase in eachindividual cell. The reaction can be stopped by addition of acompetitive inhibitor, phenylethyl-β-D-thiogalactopyranoside (PETG),making the timing of the reactions more convenient. Propidium iodidestaining is used to detect cells which have been lysed (dead cells). Theinhibitor, chloroquine, is available to lower lysosomal pH level andprevent nonspecific lysosomal hydrolysis.

FACS analysis. Cells are resuspended in 10 ml of PBS with 1% BSA and insome cases 0.1% sodium azide. Cell counts are done and viabilitiesrecorded. Cell concentrations are adjusted to 1×10⁶/ml. A 1 ml aliquotis placed in each tube and cells are spun-down. Cells are incubated withthe appropriate dilution of antibodies in 50 μl at 4° C. or at roomtemperature for 30 min. Cells are then spun and washed 2× with 1 ml ofPBS with 1% BSA and 0.1% sodium azide. The pellet of the last wash isthen incubated with an secondary reagent if appropriate, and incubatedfurther at 4° C. or at room temperature for 30 min. Cells are then spunand washed 2× with 1 ml of PBS with 1% BSA and 0.1% sodium azide. Afterthe last wash the cells are resuspended in 300 μl of PBS with 1% BSA and0.1% sodium azide and 300 μl of 2% paraformaldehyde, to fix them, priorto FACS analysis in an FACScan, Becton Dickinson, Lincoln Park, N.J.

Preparation of the Complex and Transduction of Non-adherent Cells. Theviral vector complex was prepared by mixing about 10 μl each ofbiotinylated viral particles, antibody and SA-PA fusion protein, eachhaving approximately 0.3 mg/ml of protein, in a microcentrifuge tube.The mixture was incubated for 10 minutes at 22° C. Thereafter, 2×10⁶cells were suspended in 1 ml of fresh complete medium and incubated fourhours at 37° C. in a CO₂ incubator. Thereafter, the cells were fed withan additional 4 mls of medium and cultured until FACS analysis at 48hours post transduction.

6.2.2. Transduction of Ex Vivo Target Cells with βgal

To detect infection by ecotropic viruses, we used a 48 hour assayinvolving the lacZ gene which encodes a β-galactosidase. No selectionprocess was required in this assay and good quantitation can be achievedby the fluoreporter lacZ flow cytometry assay for the detection of lacZencoded β-galactosidase in single cells (Fiering, S. N., et al., 1991,Cytometry 12:291-301; Nolan, G. P., et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:2603-2607; Molecular Probes, Inc.).

In this assay, only cells with fluorescence greater than a predeterminedlevel are considered infected. The predetermined level varied from cellto cell type. A small number of cells in the uninfected population wereabove the selected fluorescence level and these represented thebackground level of the assay.

ψ-2BAG, a murine ecotropic virus, was unable to infect a human Blymphoblastoid cell line, Daudi. However, when this virus wasbiotinylated and used in conjunction with the Streptavidin/ Protein A(SA-PA) fusion protein and either of two mAbs specific for exposedacceptors on these cells (anti-HLA-DR and anti-CALLA mAbs), infection ofDaudi cells is readily achieved, as is shown below.

TABLE I Transduction of Daudi Cells ψ-2BAG SA-PA Antibody % β galstaining No No None 2.8 Yes No None 4.8 Yes Yes anti-HLA-DR 31.6 Yes Yesanti-CALLA 24.4 Yes Yes anti-CD4 5.2

Daudi cells were not transduced when a mAb (anti-CD4) was used that didnot bind to Daudi cells. Since Daudi cells are not transduced by ψ-2BAG,these results demonstrate that the use of a vector complex comprising amurine ecotropic virus having a lacZ gene as the gene of interest, andan adapter comprising biotin, a SA-PA fusion protein and mAbs specificfor a target cell receptor was operable. It showed infection of specifichuman cells, by murine ecotropic retroviruses.

A variety of cell lines were tested using biotinylated ψ-2BAG, targetedby specific mAbs and the SA-PA fusion protein are shown in Table II,below. Of four cell lines tested SupT showed the highest rate ofinfection. These data demonstrated that the invention can besuccessfully used for various cell lines. Thus JURKAT cells was infectedby the FP-based approach, whether CD4 or CD3 molecules were targeted andCCL119 was infected by targeting CD4, CD3, or CD10.

FIG. 1 is a histogram of the fluorescence intensity of SupT withouttransduction and after transduction with the above-described viralvector complex.

TABLE II Transduction of Various Cell Types Cell line Antibody %  %Cont.⁺ KG-1 CD34 1.8 0.9 Molt-4 CD4 22.4 1.2 Jurkat CD3 6.8 1.5 JurkatCD4 4.4 1.5 CCL119 CD3 5.9 1.8 CCL119 CD4 5 1.8 CCL119 CD10 10 1.8 SupT1CD4 95 7.8 SupT1 CD4 93** 7.8 *The percent of transduced cells withβ-galactosidase dependent staining greater than threshold.

6.2.3. Transduction of In Vivo Targets by Vector Complexes

To demonstrate the operability of the invention in vivo nude mice wereinjected with human Daudi cells, and the tumor cells allowed to grow forabout 4 weeks. At this point mice were injected intraperitoneally andintratumorally with a vector complex comprising a β-gal containingbiotinylated ecotropic retroviral particle, linked to aanti-HLA-DR-PASA. Two days later the tumor cells were removed andexamined by double fluorescence for β-galactosidase activity andpresence of HLA-DR determinants. Although, in theory, the ecotropicvirus might have been infectious for the host (murine) cells, previousexperiments had shown that the biotinylated viral particles werenon-infectious in the absence of an adapter. The results of thisexperiment were that 77.3% of the recovered cells were HLA-DR positive,10.2% β-galactosidase positive, and 8.6% positive for both β-gal andHLA-DR. Thus, the bulk of β-gal positive cells were HLA-DR positive(83%).

The interpretation of the following experiment depends upon theinability of the biotinylated vectors, without adapters, to infectmurine cells. CD4 murine cells were targeted by the intravenousadministration into mice of a viral complex having an adapter consistingof a biotin on an envelope protein of the ecotropic viral particle,various concentrations. Control mice were infected only with individualcomponents of the complex. Several days after injection of the fusionprotein, animals were sacrificed and assayed for fluorescence intensityin the T and B cell populations of various organs (i.e., thymus, spleenand mesenteric lymph nodes). A comparative enrichment of theβ-galactosidase activity was seen in CD4 populations in organs of micereceiving the anti-CD4-FP-V complex. Representative results are shown inFIG. 2. In this figure the mean fluorescence intensity of the doublepositive population (CD4/β-gal) ranged from 138.22 to 163.94 in organstaken from control mice. However, mean fluorescence intensity forCD4/β-gal positive cells increased to anywhere from 326.50 to 452.17 forlymphocytes taken from animals receiving the complete vector complex. Bycontrast non-targeted B (B220 positive) cells did not demonstrate anincrease in β-gal mean fluorescence intensity in any of the animals.Double positive B220/β-gal demonstrated a mean fluorescence intensityranging from 117.31 to 138.96 in control mice and between 131 and 137.04in animals receiving the complex.

6.2.4. A Linking Molecule with Non-Antibody Acceptor Binder

The adapter comprising a fragment of containing residues 16-133 ofstreptavidin, and residues 1-50 of TAGα was constructed. The fusionprotein is expressed by the methods as described hereinabove concerningthe SA-PA fusion protein. The plasmid PTSALA, which can used to expressthe TAGα-SA fusion protein, has been deposited on Dec. 22, 1994 as ATCCNo. 69729.

6.3. Selection of the Acceptor on the Target Cell

In the preferred embodiment of the invention, the binding of the vectorcomplex to the acceptor molecule on the target cell will be followeddirectly by the internalization of the complex. The suitability of aparticular differentiation antigen can be determined by observing therate of internalization of the of the complex. The rate ofinternalization can be determined by staining with afluorescence-labeled antibody specific for an element of the adapter,typically an anti-immunoglobulin antibody. The time course ofinternalization of a complex of biotinylated viral particles, SA-PAfusion proteins and a monoclonal antibody was observed for complexescomprising anti-CD4 on SupT1 cells and anti-HLA-DR on Daudi cells. Theresults are shown in FIG. 3.

We also examined the relationship between the fusogenic activity of thevirus and transduction by the vector complex. Dextran sulfate has beenshown to inhibit viral-mediated fusion. Dextran sulfate (DS) inhibitedthe transduction of Daudi cells with β-gal mediated by an anti-HLA-DRmonoclonal antibody containing vector complex: Control 21% β-galpositive; 2 μg DS/ml, 10% positive; 6 μg DS/ml, 6% positive. Thisobservation leads one to conclude that the fusogenic activity of thevirus plays an important part of the mechanism of entry of retrovirionsmediated by FP complexes.

6.4. An Adapter that Comprises an Envelope/Streptavidin Fusion Protein

6.4.1. Construction of the pST-env Plasmid

The plasmid pST-env was constructed as follows. Total AKV RNA wasisolated from NIH3T3 cell line infected with a clonal isolate ofecotropic AKV murine retrovirus; total viral RNA consisted of atranscript that contained the entire genome and of a spliced transcriptthat contained only the env gene. The transcripts were reversetranscribed using Superscript RNase H-reverse transcriptase (LifeTechnologies, Inc., Gaithersburg, Md.) and random hexamer primers (NewEngland Biolabs, Beverly Mass.). Based on published genomic sequences ofmurine retrovirus AKV, primers were designed to the unique 5′ region(FP1: 5′-GG ACTAGT TCC GAA TCG TGG TCT CGC TGA-3′ SEQ ID NO: 1) and thecarboxyl terminus of the signal peptide of the env gene (RP1: 5′-GGG AATTC CATATG GGG GTT GAC CCC TCC GAG-3′ SEQ ID NO:2). Restriction enzymesequences for SpeI and NdeI which were engineered into the primers FP1and RP1, respectively, are in italics and irregularly grouped.

A 507 bp portion of the env gene (containing the U5 and the signalpeptide of envelope protein) was amplified by PCR (Perkin-Cetus Corpn.,Norwalk, Conn.) using FP1 and RP1 as the primers. This amplifiedfragment was cloned directly into the TA cloning vector PCR II(Invitrogen Corp., San Diego, Calif.) between the two EcoRI sites, andchecked by restriction enzyme digestion and sequence. The vector PCR IIwith the correct insert was digested with NdeI and KpnI, creating aNdeI-KpnI fragment of approximately 4.4 Kb length. The plasmid pTSAPA-2(obtained as in section 6.2.1 containing an insert encoding astreptavidin-protein-A chimeric protein was digested with the enzymesNdeI and KpnI. A resulting 360 bp fragment containing the truncatedstreptavidin was legated into the cut vector PCR E which contained theU5+ leader peptide. One clone, PCRII-10b4ST, which has the 862 bp ofcorrect insert was obtained and digested with SpeI and EcoRI to releasethe fragment of interest.

The AKV env gene (cloned into the SmaI site of the vector pBKCMV) (Lenz,S. et al., 1982, J. Virol. 49:471-8) was digested with BamI-H andHindIII. This gives a 1322 bp fragment which encodes the proline richregion of the AKV gp70 and includes the complete p15E. The BamHI-HindIIIfragment was ligated with the SpeI-EcoRI fragment with the aid oflinkers EB1 (5′-AAT TCG GGA GGC GGT GGA TCA GGT GGA GGC GGT TCA GG-3 SEQID NO: 3)′ and EB2 (5′ -GAT CCC TGA ACC GCC TCC ACC TGA TCC ACC GCC TCCCG-3′ SEQ ID NO: 4) which were compatible with the ends.

The insert encoding streptavidin and the BamHI-HindIII fragment of AKVenv was cloned into pCEP4 eukaryotic expression vectors which has ahygromycin resistance gene, and chimeric envelope construct pST-env wasobtained. A schematic representation of which is presented in FIG. 4.

Streptavidin containing fusion proteins, which have either theNspI-HindII fragment of the AKV env gene (nts 539-2191), encodingvariable region A (vrA) of the gp70 and a p15E or the Esp3I-KpnIfragment of the env gene (nts. 871-2191) that encodes only p15E havebeen made. These fusion proteins are expressed in packaging cell linesand the portion of the fusion protein derived from env is sufficient tooperable to direct the assembly of the fusion protein into the viralparticles made by the transfected packaging cell line. The plasmidpST-env was deposited on Dec. 22, 1994 as ATCC No. 69730.

6.4.2. Transfection of a Packaging Cell Line

pST-env was transfected to ψ-2BAGα ecotropic-MLV packaging cell line byCaPO₄ and lipofectin methods. First, streptavidin expression on the cellsurface was examined by using biotinylated IgG and ¹²⁵I-Protein A 3 daysafter transfection. pST-env transfected ψ-2BAGα cells showed higherbiotin binding activity than AKV env transfected control. Severaltransfected clones were picked up after hygromycin selection andexamined for its streptavidin expression by FACS analysis by usingFITC-biotin X. Some clones showed monophasic shift in fluorescenceindicating the expression of streptavidin on the cell surface.

Virus-containing supernatants were harvested from subclones, that weregrown to confluence after overnight incubation in serum-freenonselective medium. Virus supernatants were incubated with biotinylatedanti-HLA-DR mAb for 30 min at RT then added to Burkitt's lymphoma Daudicells and incubated for 4 hr at 37° C. Infected cells were examined bylacz-staining flow cytometry (Molecular Probe, Inc.) 3 days afterinfection. Negative control infections were done with virus similarlyharvested from ψ-2BAGα cells. As shown by the comparison of FIGS. 5A and5B, 20% of Daudi cells transduced with a pST-env-transfected cell linesupernatant contained β-gal related staining; control Daudi cellstransduced with unmodified ψ-2BAGα supernatants had a level of only 2.7%ψ-gal related staining. This result demonstrated the production of anecotropic-MLV which contains streptavidin-env fusion protein adapter.

The present invention is not to be limited in scope by the specificembodiments described which were intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components were within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

4 29 base pairs nucleic acid single linear DNA 1 GGACTAGTTC CGAATCGTGGTCTCGCTGA 29 32 base pairs nucleic acid single linear DNA 2 GGGAATTCCATATGGGGGTT GACCCCTCCG AG 32 38 base pairs nucleic acid single linear DNA3 AATTCGGGAG GCGGTGGATC AGGTGGAGGC GGTTCAGG 38 38 base pairs nucleicacid single linear DNA 4 GATCCCTGAA CCGCCTCCAC CTGATCCACC GCCTCCCG 38

We claim:
 1. A viral vector complex for transducing a target cell with agene of interest under physiological conditions, comprising: a. a viralparticle, comprising
 1. a gene of interest operably linked to a promoterthat is active in a target cell;
 2. an envelope protein;
 3. a biotinmolecule covalently affixed to said envelope protein; and b. abifunctional adapter affixed to said envelope protein, the bifunctionaladapter comprising a binding site for a native acceptor on the targetcell and a linking molecule comprising a biotin binding site and abinding site for the native acceptor, wherein the linking molecule isnon-covalently bonded to said envelope protein.
 2. The viral vectorcomplex of claim 1 wherein the binding site for the native acceptor is abinding site for a carbohydrate.
 3. The viral vector complex of claim 1which the acceptor binding site is a fragment of a soluble cytokineselected from the group consisting of erythropoietin, interleukins,interferons, fibroblast growth factors, transforming growth factors,tumor necrosis factors, colony stimulating factors and epidermal growthfactor.
 4. The viral vector complex of claim 1 in which the linkingmolecule comprises a Protein A/Streptavidin fusion protein and anantibody molecule that binds to the native acceptor.
 5. The viral vectorcomplex of claim 4 in which the native acceptor is selected from thegroup consisting of class I MHC antigens, class II MHC antigens,internalizing cell-surface receptors and viral receptors and the bindingsite for a native acceptor is a binding site for a protein portion ofthe native acceptor.
 6. A method of expressing a gene of interest in atarget cell which comprises directly contacting the target cell with theviral vector complex of claim 1 under physiological conditions whereinthe viral particle is internalized into the cell so that the cell istransduced and expresses the gene of interest.
 7. The method of claim 6wherein the target cell is a target cell cultured ex vivo.
 8. The methodof claim 6 wherein the target cell is a target cell present in amammalian animal.
 9. A method for preparing a viral vector complex fortransducing a target cell with a gene of interest under physiologicalconditions, comprising: (a) contacting under conditions suitable fornon-covalent bond formation: (i) a viral particle having a gene ofinterest operably linked to a promoter which is active in the targetcell, and an envelope protein, wherein a biotin molecule is covalentlyaffixed to said envelope protein, with (ii) a bifunctional adaptermolecule comprising a binding site for a native acceptor of the targetcell and a linking molecule that is capable of non-covalent bonding tosaid viral envelope protein wherein the linking molecule comprises abiotin binding site and a binding site of the native acceptor, andbonded to the viral envelope protein.
 10. The method of claims 9 whereinthe envelope protein is a viral envelope protein.